Mechanistic Design of Polymer Nanocarriers to Spatiotemporally Control Gene Silencing

Current siRNA delivery systems lack the ability to precisely tune siRNA release and maximize gene silencing in a spatiotemporal manner. Herein, we investigate photoresponsive block copolymer solution assemblies, for which stimuli-triggered changes in polymer structure altered nanocarrier stability and defined siRNA activity. Uniquely, our biomaterials design enabled the development and validation of a simple kinetic model that accurately predicted the extent of intracellular nanocarrier disassembly and silencing. Moreover, our constructs showed that maximal gene silencing could be achieved using concentrations of siRNA 5-fold lower than typical formulations due to the ability to rapidly release sufficient amounts of siRNA to saturate the cellular RISC machinery. The ability of our nanocarriers to remain dormant prior to phototriggered siRNA release allowed for the generation of cell patterns in gene expression with spatial control on cellular length scales and no detectable off-target effects. Furthermore, precisely tuned changes in nanocarrier structure enabled the modulation of protein and mRNA knockdown levels in murine fibroblasts and terminally differentiated human primary cells. These advances lead to increased precision, potency, and utility relative to other recent spatiotemporally controlled nucleic acid delivery vehicles reported in the literature. Moreover, the combination of experimental examination and kinetic modeling described herein should be applicable to a host of systems for which temporal control over nucleic acid delivery is a critical parameter in influencing cellular responses.